We have isolated pure Chinese hamster cell ribosomal subunits, extracted their proteins, and analyzed 72 ribosomal proteins by two-dimensional gel electrophoresis. Thirty-two of the proteins were associated with the 40s subunits and 40 proteins were associated with 60s subunits. We have attempted to isolate Chinese hamster cell clones resistant to cycloheximide, anisomycin and emetine and have obtained a collection of 9 mutants resistant to emetine. In one emetine-resistant mutant, Emr-2, we detected as single altered 40S ribosomal subunit protein, S20. None of the proteins of Emr-2's 60S subunit nor of the other eight mutants' ribosomes were altered by mutation sufficiently to be detected by electrophoresis. Therefore, most ribosomal mutations isolated in tissue culture have involved conservative amino acid changes. To determine whether altered s20 is responsible for the Emr phenotype, we have constructed tetraploid Emr-2 X wild-type CHL cell hybrids (Emr/Ems) and have isolated several Emr mitotic segregants from each hybrid. All of the hybrids are heterozygous for S20 protein, as they expressed and assembled into all segregants tested so far expressed only the Emr-2 S20. Thus, the S20 gene, or a gene closely linked to it, is responsible for emetine-resistance in Chinese hamster cells.